Inhibition of thiaminase I from Bacillus thiaminolyticus. Evidence supporting a covalent 1,6-dihydropyrimidinyl-enzyme intermediate

Abstract

Thiaminase I from Bacillus thiaminolyticus strain Matsukawa et Misawa is completely and irreversibly inhibited by treatment with 4-amino-6-chloro-2-methylpyrimidine. Inhibition is a time-dependent first-order process, exhibiting a half-time of 4 h at an inhibitor concentration of 5 mM. A specific active-site-directed inactivation is supported by protection of the enzymatic activity in the presence of the substrates thiamin and quinoline as well as by the observation that a stoichiometric amount of inorganic chloride is released during inactivation. 4-Amino-5-(anilinomethyl)-6-chloro-2-methylpyrimidine, which resembles the structure of the product of base exchange of thiamin with aniline, inactivates thiaminase approximately 2 orders of magnitude faster. Inactivation is again complete and irreversible and is a time-dependent first-order process, in this case exhibiting saturation at low inhibitor concentrations (KI = 96 microM). Enzyme inactivation can be explained as the result of displacement of chloride from the chloropyrimidine by a nucleophile at the enzyme active site. The inactivation suggests that the Zoltewicz-Kauffman model of bisulfite-catalyzed thiamin cleavage [Zoltewicz, J. A., & Kauffman, G. M. (1977) J. Am. Chem. Soc. 99, 3134-3142], which calls for the reversible nucleophilic addition of catalyst across the 1,6 double bond of thiamin's pyrimidine ring, may be applicable to thiaminase as well.

Identification of Modification Sites in Large Biomolecules by Stable Isotope Labeling and Tandem High Resolution Mass Spectrometry

“…Incubation with the suicide substrate 2 (18,22) increased their masses by 108 Ϯ 1 Da, consistent with the 107-Da mass difference (⌬m) expected for Cl Ϫ release during inactivation of one active site in each of the three enzyme molecules. This spectrum showed the isotopic cluster around 42,198 Da to be Ͻ5% as abundant as that around 42,305 Da (16), indicating that derivatization was Ͼ95% complete, as confirmed by 2-nitro-5-thiol sulfobenzoate thiol titration (18).…”

“…Synthesis-4-Amino-6-chloro-2,5-dimethylpyrimidine (2-CH 3 ) was synthesized from diethyl methyl malonate in a three-step sequence using the route previously described for the synthesis of 2 (18,22). The product was purified by two stages of silica gel chromatography (90:10 chloroform/methanol, 80:20 ethyl acetate/hexanes).…”

Mechanistic Studies on Thiaminase I

“…From these studies, and the inactivation of the enzyme with 2-methyl-4-amino-6-chloropyrimidine, a mechanism involving the addition of an active site nucleophile to C 6 of the pyrimidine has been proposed ( Fig. 10) (Hutter and Slama, 1987;Lienhard, 1970). This mechanistic proposal is also supported by extensive studies on the mechanism of thiamin cleavage with bisulfite (Uray et al, 1993).…”

“…Active Site Analysis of Thiaminase I-Thiaminase I was inactivated (K I ϭ 7.7 mM, k inact ϭ 0.7 h Ϫ1 ) by treatment with 2-methyl-4-amino-6-chloropyrimidine (8, Hutter and Slama, 1987). ESI/FT-MS analysis of the inactivated enzyme indicated that all three species were labeled with the pyrimidine moiety and that chloride was lost during the inactivation (⌬molecular mass ϭ ϩ108, Fig.…”

Structure of a eukaryotic thiaminase I

“…The base substitution reaction of thiamin by thiaminase I is proposed to proceed via a double addition-elimination mechanism based on several observations made with the enzyme from B. thiaminolyticus: the reaction proceeds via ping-pong kinetics (12,13,35); the substitution reaction using chiral monodeuteriothiamin proceeds with retention of stereochemistry (36,37); and by the mechanism-based inactivation of the enzyme with Pyd resulting in a Cys113 covalent adduct (31,38). Given the similarity between Bt-thiaminase and Ng-thiaminase, the expectation is that the mechanism should be similar if not exactly the same.…”